Mesenchymal stem cells from rodent and human bone marrow (BM-MSC) can differentiate into muscle, bone, cartilage, fat, and fibroblast. We have recently characterized human cells with a similar differentiative capacity, which are isolated from a more accessible and plentiful source; human adipose tissue. These human adipose-derived mesenchymal stem cells (AMSC) expand rapidly in culture, are easily transduced by retroviral vectors, and home into multiple tissues in immune deficient mice, including brain. However, like BM-MSC, the phenotype of the most primitive cells in the AMSC population is not known, and the rapidly growing plates of cells that we have studied in vitro and in vivo are likely quite heterogeneous. A major goal of the proposed studies is to phenotypically characterize and to define the function of the most primitive populations of AMSC and BM-MSC, in vitro and in vivo. We have identified a subpopulation of cells in both BM-MSC and AMSC that expresses low levels of c-met, the receptor for Hepatocyte Growth Factor (HGF). HGF is a chemoattractant and viability factor that is upregulated at the site of injury in many tissues, including muscle, liver, intestine, and lung, plus cardiac and skeletal muscle. HGF affects the motility and maintains the viability of many primitive cell types. We hypothesize that HGF, secreted locally in response to tissue injury, is a major factor in recruiting stem cells from the circulation into the site of injury to mediate repair, and that the most primitive and pluripotent MSC will express c-met, the receptor for HCF. We will therefore begin our phenotypic dissection of the AMSC and BM-MSC compartments by isolating human C-met+/CD45- cells from adipose tissue and bone marrow. We will further subfractionate the human CD45-/e-met+ and CD45-/c-met- populations into subsets that do and do not express CD105, and CD133. In Specific Aim 1, we will test the candidate pluripotent MSC populations for their capacity to differentiate to muscle, bone, cartilage, and fat in vitro to demonstrate their multipotency. In Specific Aim 2, we will examine the in vivo homing of the AMSC and BM-MSC subpopulations into specific tissues of immune deficient mice, during chronic and acute injury states in muscle, pancreas, liver, and brain. In this aim, we will specifically seek populations of human MSC that can not only home into the injured murine tissues, but those that can also mediate repair in a robust manner. Finally, in Specific Aim 3, we will induce the candidate pluripotent human MSC populations into cycle in vitro, mark them with retroviral vectors, and perform clonal integration analysis studies following in vitro and in vivo differentiation. This final aim will stringently identify the subpopulations of human MSC from bone marrow and adipose tissue which can differentiate into progeny of multiple lineages. The overall goal of the proposed studies is to systematically dissect the hierarchy of human MSC differentiation and to develop models to analyze the performance of each subpopulation in tissue repair so that these promising cells can be best harnessed for safe and rational regenerative therapies.